CN100567501C - Method for producing biological substances in pigment-deficient mutants of bacillus cells - Google Patents

Abstract

The present invention relates to a method of producing heterologous biological substances, comprising: culturing a mutant of a parent bacillus cell under conditions conducive for production of the heterologous biological substance, wherein (i) the mutant cell contains a first nucleic acid sequence that directs synthesis of the heterologous biological substance and a second nucleic acid sequence that comprises a modification in at least one of the cypX and yvmC genes involved in red pigment production, and (ii) the mutant cell is deficient in red pigment production as compared to the parent bacillus cell cultured under the same conditions; and (b) recovering the heterologous biological substance from the culture medium. The invention also relates to mutants of bacillus cells and methods of producing the mutants.

Description

Method for producing biological material in pigment deficient mutants of Bacillus cells

Background of the invention

field of invention

The present invention relates to methods of producing heterologous biological substances in pigment-deficient mutants of Bacillus cells, methods of obtaining pigment-deficient mutants of Bacillus cells, and pigment-deficient mutants of Bacillus cells.

Description of related technologies

Pulcherrimin is a light red pigment formed by the chelation of pulcherriminic acid and iron ions. Purchemin consists of a substituted pyrazine ring incorporating an isobutyl group at positions 2 and 5, but other structural details differ slightly (Kuffer et al., 1967, Archiv für Mikrobiologic 56:9-21).

MacDonald, 1967, Canadian Journal of Microbiology 13: 17-20, has described the isolation and conversion of prucemin from Bacillus cereus and Bacillus subtilis to the free acid chimeric acid. Uffen and Canale-Parola, 1972, Journal of Bacteriology 111: 86-93, describe the synthesis of chimeric acid by Bacillus subtilis.

Bacillus is a well-established host cell system for the production of natural and recombinant proteins or other biological substances. However, a Bacillus host having the desired properties of increased expression and secretion of proteins may not necessarily have the most desirable properties for successful fermentation, recovery and purification of the biomass produced by the cells. These processes may not be optimal because pigment formation needs to be removed during recovery and purification of the target biological material or the pigment can co-purify with the biological material.

It is therefore an object of the present invention to provide improved Bacillus hosts which have both the ability to express commercial quantities of biological material and which are deficient in red pigment production.

Summary of the invention

The present invention relates to methods of producing heterologous biological substances, comprising:

(a) culturing a mutant of the parental Bacillus cell under conditions suitable for the production of heterologous biological material, wherein (i) the mutant cell contains a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence directing Synthesis of heterologous biological substances, the second nucleic acid sequence comprising a modification in at least one of the cypX and yvmC genes involved in red pigment production, and (ii) the mutant cells are the same as the parental Bacillus cells cultured under the same conditions In contrast, red pigment production is defective; and

(b) recovering said heterologous biological material from said culture medium.

The present invention also relates to red pigment-deficient mutants of Bacillus cells and methods of producing red pigment-deficient mutants of Bacillus cells.

Description of drawings

Figures 1A and 1B show the genomic DNA sequence of the cypX gene and its deduced amino acid sequence (SEQ ID NOS: 1 and 2, respectively).

Figures 2A and 2B show the genomic DNA sequence of the yvmA gene and its deduced amino acid sequence (SEQ ID NOS: 3 and 4, respectively).

Figure 3 shows the genomic DNA sequence of the yvmB gene and its deduced amino acid sequence (SEQ ID NOS: 5 and 6, respectively).

Figures 4A and 4B show the genomic DNA sequence of the yvmC gene and its deduced amino acid sequence (SEQ ID NOS: 7 and 8, respectively).

Fig. 5 is a restriction enzyme map of pMRT084.

Fig. 6 is a restriction enzyme map of pMRT086.

Fig. 7 is a restriction enzyme map of pMRT126.

Fig. 8 is a restriction enzyme map of pMRT128.

Fig. 9 is a restriction enzyme map of pMRT121.

Figure 10 is a restriction enzyme map of pMRT123.

Figure 11 is a restriction enzyme map of pMRT124.

Detailed description of the invention

The present invention relates to a method for producing heterologous biological material comprising: (a) cultivating a mutant of a parental Bacillus cell under conditions conducive to the production of a heterologous biological material, wherein (i) said mutant cell contains a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence directs the synthesis of heterologous biological substances, the second nucleic acid sequence comprises modification in at least one of the cypX and yvmC genes involved in red pigment production, and (ii) the mutation somatic cells are defective in red pigment production compared to parental Bacillus cells cultured under the same conditions; and (b) recovering said heterologous biological material from said culture medium. .

The advantage of the present invention is that the red pigment is eliminated or reduced in the Bacillus fermentation broth. The elimination or reduction of the red pigment facilitates the recovery and purification of target biological substances.

In the method of the present invention, it is believed that the red pigment is prochemin, because when a solid or liquid medium of a Bacillus culture is grown in the absence of iron ions and then exposed to iron ions, the culture and/or cells turned light red. In addition, the isolated pigment is soluble in alkali, insoluble in water and organic solvents, and its ultraviolet-visible spectrum is consistent with that of the previously published polar acid spectrum (see, Canale-Parola, 1963, Archiv für Mikrobiologie 46: 414-427). The term "purchemin" is defined herein as an iron chelate or iron salt of chimeric acid. Chimeic acid is the free acid of pulcemin, which consists of a substituted pyrazine ring incorporating an isobutyl group at positions 2 and 5, differing slightly in other structural details (Kuffer et al., 1967, supra).

The term "modification" is defined herein as the introduction, substitution, or removal of one or more nucleotides in a gene or its regulatory elements required for transcription or translation; gene disruption; gene conversion; gene deletion; or the cypX and yvmC genes Random mutation or specific mutation in at least one gene. The deletion of the cypX and/or yvmC genes may be partial or complete.

The phrase "defective in red pigment production" is defined herein as a Bacillus mutant cell that does not produce a detectable red pigment, or that, compared to a parental Bacillus cell cultured under the same conditions, the mutant cell produces Red pigment is preferably at least about 25% less, more preferably at least about 50% less, even more preferably at least about 75% less, and most preferably at least about 95% less. The level of red pigment produced by the Bacillus mutant cells of the invention can be determined using methods well known in the art (see, eg, Kuffer et al., 1967, supra). However, since the pigment is adsorbed to the cells regardless of whether the medium used is complex or minimal, the presence or absence of the red pigment can be observed by centrifuging the cells. In minimal media, a red pigment can be observed in the supernatant, but as the media composition becomes more complex and stained by media components, the color of the media components can mask the red pigment in the cell supernatant presence or absence.

In the method of the present invention, the parental Bacillus cell may be a red pigment-producing wild-type Bacillus cell or a mutant thereof. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Gram Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, giant Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In a preferred embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In a more preferred embodiment, the parent Bacillus cell is a Bacillus amyloliquefaciens cell. In another more preferred embodiment, the parent Bacillus cell is a Bacillus clausii cell. In another more preferred embodiment, the parent Bacillus cell is a Bacillus licheniformis cell. In another more preferred embodiment, the parent Bacillus cell is a Bacillus subtilis cell.

The red pigment-deficient mutant of Bacillus cells can be constructed by reducing or eliminating the expression of at least one of the cypX and yvmC genes using methods known in the art, for example, insertion, disruption, substitution, or deletion. The portion of the gene that is modified or inactivated can be, for example, the coding region or the regulatory elements required for expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, ie a part sufficient to affect the expression of a nucleic acid sequence. Other control sequences that may be modified include, but are not limited to, leaders, propeptide sequences, signal sequences, transcription terminators, and transcription activators.

Bacillus mutant cells can be constructed by eliminating or reducing the expression of at least one of the cypX and yvmC genes using gene deletion techniques. Gene deletion techniques can remove part or all of the genes, thereby eliminating their expression. In this method, gene deletion can be accomplished by homologous recombination using a plasmid containing the 5' and 3' side contiguous regions of the gene. The 5' and 3' contiguous regions can be introduced into a Bacillus cell at a permissive temperature, e.g., into its temperature-sensitive plasmid (such as pE194), combined with a second selectable marker, so that the plasmid becomes in said cell Can be accepted (become established). Cells are then transferred to a non-permissive temperature, and those cells that have integrated one of the plasmids into a homologous flanking region of the chromosome are selected. Selection for plasmid integration is achieved by selection for a second selectable marker. Following integration, cells are transferred to a permissive temperature for several passages without selection, thereby stimulating recombination events at the second homologous flanking region. Cells are plated to obtain single colonies, and these colonies are checked for loss of the above two selectable markers (see, e.g., Perego, 1993, in Bacillus subtilis and Other Gram-Positive eds., A.L. Sonneshein, J.A. Hoch, and R. Losick eds. Bacteria, Chapter 42, American Society of Microbiology, Washington, D.C.).

Bacillus mutant cells can also be constructed by introducing, substituting, or removing one or more nucleotides in a gene or its regulatory elements required for transcription or translation. For example, nucleotides may be inserted or removed to introduce stop codons, remove start codons, or to frameshift the open reading frame. This modification can be accomplished by site-directed mutagenesis or PCR mutagenesis according to methods known in the art. See, e.g., Botstein and Shortle, 1985, Science 229:4719; Lo et al., 1985, Proceedings of the National Academy of Sciences USA 81:2285; Higuchi et al., 1988, Nucleic Acids Research 16:7351; Shimada, 1996, Meth. Mol. Biol. 57:157; Ho et al., 1989, Gene 77:61; Horton et al., 1989, Gene 77:61; and Sarkar and Sommer, 1990, Bio Techniques 8:404.

Bacillus mutant cells can also be constructed by gene disruption techniques by inserting into one or more of the genes responsible for the red pigment an integrated plasmid containing a nucleic acid segment homologous to said gene which will generate duplicate homologous regions And the carrier DNA is incorporated between the duplicate regions of homology. If the inserted vector separates the gene's promoter from the coding region, or disrupts the coding sequence so that a non-functional gene product is produced, such gene disruption can abolish gene expression. The disrupted construct may simply be the selectable marker gene with 5' and 3' regions homologous to the gene. A selectable marker enables the identification of transformants containing disrupted genes.

Bacillus mutant cells can also be constructed by genetic transformation (see, eg, Iglesias and Trautner, 1983, Molecular General Genetics 189:73-76). For example, in the gene conversion method, the nucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into a parental Bacillus cell to produce the defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. Preferably, the defective gene or gene fragment also encodes a marker that can be used to select for transformants containing the defective gene. For example, a defective gene can be introduced on a non-replicating or temperature-sensitive plasmid along with a selectable marker. Selection for plasmid integration is accomplished by selection on markers under conditions that do not allow plasmid replication. Selection for a second recombination event leading to gene replacement is accomplished by examining colonies for loss of the selectable marker and acquisition of the mutated gene (see, eg, Perego, 1993, supra). Alternatively, a defective nucleic acid sequence may contain an insertion, substitution, or deletion of one or more nucleotides of a gene, as described below.

Bacillus mutant cells can also be constructed using a nucleotide sequence complementary to that of the gene by well-established antisense technology (Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). More specifically, gene expression in Bacillus cells can be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which is transcribed in the cell and capable of hybridizing to mRNA produced in the cell. Under conditions such that the complementary antisense nucleotide sequence hybridizes to the mRNA, the amount translated into protein is thus reduced or eliminated.

Bacillus mutant cells can also be further constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis (see, e.g., Hopwood, The Isolation of Mutants in Methods in Microbiology (J..R.Norris and D.W.Ribbons edited) pp 363-433, Academic Press, New York, 1970) and transposition (see, e.g., Youngman et al., 1983, Proc.Natl.Acad.Sci.USA 80: 2305-2309). Genetic modification can be performed by mutagenizing the parental cells and selecting mutant cells for which gene expression has been reduced or eliminated. Specific or random mutagenesis can be performed, for example, using appropriate physical or chemical mutagens, using appropriate oligonucleotides, or mutagenic PCR on DNA sequences. In addition, mutagenesis can be performed by using any combination of these mutagenesis methods.

Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N '-Nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium sulfite, formic acid, and nucleotide analogues. When such reagents are used, mutagenesis is classically accomplished by culturing the parental cells to be mutagenized in the presence of a selective mutagen under appropriate conditions, selecting for mutant cells showing reduced or no expression of the gene.

As described herein, in the methods of the invention, the cypX gene or the yvmC gene, or both, of the Bacillus cells involved in the production of the red pigment may be modified. According to the protocol of Berka et al., 2002, Molecular Microbiology 43: 1331-1345, the cypX-yvmC operon was identified as a potential site involved in the formation of red pigment by Bacillus ORFs microarray (microarray) analysis. It should be understood that the term "second nucleic acid sequence" may include one or both of the cypX and yvmC genes.

In a preferred embodiment, cypX comprises at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% of SEQ ID NO: 1. % homology of nucleic acid sequences. In the most preferred embodiment, cypX comprises the nucleotide sequence of SEQ ID NO:1. In another most preferred embodiment, cypX is made up of the nucleotide sequence of SEQID NO:1.

In a preferred embodiment, yvmC comprises at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, even most preferably at least 95% of SEQ ID NO: 3 homologous nucleic acid sequences. In the most preferred embodiment, yvmC comprises the nucleotide sequence of SEQ ID NO:3. In another most preferred embodiment, yvmC is made up of the nucleotide sequence of SEQID NO:3.

For the purposes of the present invention, by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80:726-730), using LASERGENE ^™ MEGALIGN ^™ software (DNASTAR, Inc., Madison, WI), The degree of homology between two nucleic acid sequences is determined using an identity table and the following multiple sequence alignment parameters: a gap penalty of 10 and a gap length penalty of 10. Pairwise comparison parameters are: Ktuple=3, gap penalty=3, window=20.

Nucleic acid sequences homologous or complementary to nucleic acid sequences described herein involved in the production of red pigments from other sources of red pigment producing microorganisms may be used to modify the corresponding genes in selected Bacillus strains.

In a preferred embodiment, the modification of the genes involved in the production of the red pigment in the Bacillus mutant cells is not marked with a selectable marker.

Selectable marker genes can be removed by growing mutants on counter-selection media. The selectable marker gene contains repeat sequences flanking its 5' and 3' ends that will facilitate looping out of the selectable marker gene by homologous recombination when mutant cells are counter-selected. It can also be removed by homologous recombination by introducing a nucleic acid fragment containing the 5' and 3' regions of the defective gene but lacking a selectable marker gene into mutant cells, followed by selection on a counter-selection medium. selectable marker gene. By homologous recombination, the defective gene containing the selectable marker gene is replaced with a nucleic acid fragment lacking the selectable marker gene. Other methods known in the art may also be used.

It should be understood that the methods of the present invention are not limited to a particular mode of obtaining Bacillus mutant cells. Modifications of genes involved in the production of red pigments can be introduced into the parental cells at any step in the construction of the biological substance-producing cells. Preferably, the Bacillus mutant cells have been made to lack a red pigment prior to the introduction of the gene directing the synthesis of the heterologous biological material.

In another aspect of the invention, the Bacillus mutant cells may additionally contain modifications, eg, deletion or disruption of other genes that are deleterious to the production, recovery or use of biological material. In preferred embodiments, the Bacillus cells are protease-deficient cells. In a more preferred embodiment, the Bacillus cell contains a disruption or deletion of aprE and nprE. In another preferred embodiment, the Bacillus cells do not produce spores. In another more preferred embodiment, the Bacillus cell contains a disruption or deletion of spoIIAC. In another preferred embodiment, the Bacillus cell contains a disruption or deletion of one of the genes involved in surfactin synthesis, eg, srfA, srfB, srfC, and srfD. See, eg, US Patent No. 5,958,728. Other genes, for example, the amyE gene can also be disrupted or deleted which is detrimental to the production, recovery or use of biological material.

In the methods of the invention, it is preferred that the Bacillus mutant cells produce at least the same amount of biological material as the corresponding parental Bacillus cells when cultured under the same production conditions. In a more preferred embodiment, the mutant cells produce at least about 25% higher, preferably at least about 50% higher, more preferably at least about 75% higher, most preferably at least High about 100% biomass.

Using methods known in the art, the Bacillus mutant cells are cultivated in a nutrient medium suitable for production of heterologous biological material. For example, it can be cultured in shake flasks, small-scale or large-scale fermentation (including continuous, fractional) in a suitable medium and under conditions that allow the expression and/or isolation of biological substances in fermentors for laboratory or industrial use. Batch, fed-batch, or solid-state fermentation) to culture cells. The cultivation is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (eg, in catalogs of the American Type Culture Collection). Secreted biological material can be recovered directly from the culture medium.

Biological substances can be detected using methods known in the art that are specific for biological substances. These detection methods may include the use of specific antibodies, high performance liquid chromatography, capillary chromatography, formation of enzyme products, disappearance of enzyme substrates, or SDS-PAGE. For example, enzyme activity can be determined using an enzyme assay. Methods for measuring enzymatic activity are known in the art for many enzymes (see, eg, D. Schomburg and M. Salzmann (eds.), Enzyme Handbook, Springer-Verlag, New York, 1990).

The resulting biological material can be isolated by methods known in the art. For example, the polypeptide of interest can be isolated from the culture medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The isolated polypeptide can then be further purified by various methods known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatographic aggregation, and size exclusion chromatography), electrophoretic methods (e.g., preparative isoelectric focusing (IEF)), differential dissolution (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Publishers, New York, 1989). Metabolites of interest can be isolated from the culture medium, for example, by extraction, precipitation, or differential solubilization, or any method known in the art. The isolated metabolites can then be further purified using metabolite-appropriate methods.

A heterologous biological substance can be any biological polymer or metabolite. Biological substances can be encoded by a single gene or a series of genes that make up a biosynthetic or metabolic pathway. Accordingly, the term "first nucleic acid sequence directing the synthesis of heterologous biological material" should be understood to include one or more genes involved in the production of biological material. The term "heterologous biological material" is defined herein as biological material not produced by the host cell; natural biological material in which structural modifications have been made to alter the native biological material, for example, by altering the protein sequence of a native polypeptide; Treatment of host cells, for example, with stronger promoters, leads to altered expression of native biological substances.

In the methods of the present invention, the biopolymer can be any biopolymer. The term "biopolymer" is defined herein as a chain (or polymer) of identical, similar or dissimilar subunits (monomers). Biopolymers can be, but are not limited to, nucleic acids, polyamines, polyols, polypeptides (or polyamides), or polysaccharides.

In preferred embodiments, the biopolymer is a polypeptide. The polypeptide can be any polypeptide having the biological activity of interest. The term "polypeptide" is not meant herein to refer to a specific length of the encoded product, thus, the term includes peptides, oligopeptides and proteins. The term "polypeptide" also includes two or more polypeptides that combine to form the encoded product. Polypeptides also include hybrid polypeptides, which comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides, one or more of which may be heterologous to the Bacillus cell. Polypeptides further include natural allelic variations and genetically engineered variations of the aforementioned polypeptides and hybrid polypeptides.

Preferably, the heterologous polypeptide is an antibody, antigen, antimicrobial peptide, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, or transcription factor.

In preferred embodiments, the heterologous polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In a more preferred embodiment, the polypeptide is an alpha-glucosidase, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin, Glycosyltransferase, DNase, esterase, α-galactosidase, β-galactosidase, glucoamylase, glucocerebrosidase, α-glucosidase, β-glucosidase , invertase, laccase, lipase, mannosidase, mutant enzyme, oxidase, pectin degrading enzyme, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonucleic acid enzyme, transglutaminase, urokinase, or xylanase.

In preferred embodiments, the biopolymer is a polysaccharide. The polysaccharide can be any polysaccharide, including, but not limited to, mucopolysaccharides (eg, heparin and hyaluronic acid), hyaluronan, and nitrogen-containing polysaccharides (eg, chitin). In a more preferred embodiment, the polysaccharide is hyaluronic acid.

In the methods of the invention, the metabolite can be any metabolite. Metabolites can be encoded by one or more genes. The term "metabolite" includes primary metabolites and secondary metabolites. Primary metabolites are the products of the primary or general metabolism of the cell, which are involved in energy metabolism, growth, and structure. Secondary metabolites are the products of secondary metabolism (see, eg, R.B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981).

Primary metabolites can be, but are not limited to, amino acids, fatty acids, nucleosides, nucleotides, sugars, triglycerides, or vitamins.

Secondary metabolites can be, but are not limited to, alkaloids, coumarins, flavonoids, polyketides, quinines, steroids, or terpenes. In preferred embodiments, the secondary metabolite is an antibiotic, antifeedant, attractant, bactericide, fungicide, hormone, insecticide, or rodenticide.

In the methods of the invention, mutants of Bacillus cells are recombinant cells containing nucleic acid sequences directing the synthesis of heterologous biological substances, eg, polypeptides, which facilitate their use in the recombinant production of biological substances. Cells are preferably transformed with a vector containing a nucleic acid sequence directing the synthesis of heterologous biological material, followed by integration of the vector into the chromosome. "Transformation" means introducing a vector containing a nucleic acid sequence into a host cell, maintaining the vector as an integral part of a chromosome or as a self-replicating extrachromosomal vector. Integration is generally considered an advantage, since nucleic acid sequences are more likely to be stably maintained within the cell. The vector is integrated into the chromosome by homologous recombination, non-homologous recombination, or transposition.

Nucleic acid sequences directing the synthesis of heterologous biological material can be obtained from any prokaryotic, eukaryotic, or other source, eg, archaea. For the purposes of the present invention, the term "obtained" as used herein in relation to a given source means that biological material is produced by the source or by cells into which the genes of the source have been inserted.

In the methods of the present invention, mutants of Bacillus cells can be used to recombinantly produce the native biomass of Bacillus cells. Natural substances can be produced by recombinant methods, for example, by placing genes directing the synthesis of biological substances under the control of different promoters to increase the expression of substances, by using, for example, signal sequences, to accelerate their transport outside the cell, or by increasing the number of directing spores The number of copies of genes for the synthesis of biological material normally produced by the bacillus cell. Thus, within the scope of the term "heterologous biological material", the present invention also includes such recombinant production of natural biological material to the extent that such expression involves the use of genetic elements not belonging to Bacillus cells, or the use of manipulated , a natural element that functions in a manner that is not normally found in the host cell.

Techniques for isolating or cloning nucleic acid sequences directing the synthesis of biological substances are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Cloning of nucleic acid sequences from such genomic DNA can be performed, for example, by using the well-known polymerase chain reaction (PCR). See, eg, Innis et al., 1990, PCR Protocols: A Guide to Methods and Applications. n, Academic Press, New York. The cloning process may involve excision and isolation of the desired nucleic acid fragment containing the nucleic acid sequence directing the synthesis of biological material, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into Bacillus cells that will replicate multiple copies or clones of the nucleic acid sequence. The nucleic acid sequence may be genomic sequence, cDNA sequence, RNA sequence, semi-synthetic sequence, synthetic origin sequence, or any combination thereof.

In the methods of the invention, the biological substance is a heterologous polypeptide, such polypeptides may also include fusion polypeptides, wherein another polypeptide is fused to the N-terminus or C-terminus of a polypeptide or a fragment thereof. Fusion polypeptides are produced by fusing a nucleic acid sequence (or portion thereof) encoding one polypeptide to a nucleic acid sequence (or portion thereof) encoding another polypeptide. Techniques for producing fusion polypeptides are known in the prior art, including linking the coding sequences encoding the polypeptides so that they are in frame and expressing the fusion polypeptides under the control of the same promoter and terminator.

A "nucleic acid construct" is defined herein as a nucleic acid molecule, single or double stranded, which is isolated from a naturally occurring gene, or which has been modified to contain nucleic acid segments combined and juxtaposed differently than they occur in nature. The term nucleic acid construct may have the same meaning as the term expression cassette when the nucleic acid construct contains all the regulatory sequences required for the expression of a coding sequence. The term "coding sequence" is defined herein as a sequence that is transcribed into mRNA and translated into a biological substance of interest when it is placed under the control of the regulatory sequences described below. The boundaries of the coding sequence are generally defined by a translation initiation codon ATG at the 5'-terminus and a translation termination codon at the 3'-terminus. A coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence directing the synthesis of a biological material can be manipulated in various ways to provide for the expression of the biological material. Depending on the expression vector or Bacillus host cell, manipulation of the nucleic acid sequence may be desired or necessary prior to its insertion into the vector. Techniques for modifying nucleic acid sequences using cloning methods are well known in the art.

Under conditions compatible with the regulatory sequences, a nucleic acid construct comprising a nucleic acid sequence directing the synthesis of biological material can be operably linked to one or more regulatory sequences capable of directing the expression of the coding sequence in a Bacillus cell mutant .

The term "regulatory sequence" is defined herein to include all elements necessary for or facilitating the expression of the coding sequence of a nucleic acid sequence. Each regulatory sequence may be native or heterologous to the nucleic acid sequence directing the synthesis of biological material. These regulatory sequences include, but are not limited to, leaders, promoters, signal sequences, and transcription terminators. At a minimum, regulatory sequences include a promoter, and transcriptional and translational stop signals. The regulatory sequence can be provided with a linker for the purpose of introducing a specific restriction enzyme site, so as to facilitate the connection of the regulatory sequence and the coding region of the nucleic acid sequence directing the synthesis of biological substances.

The control sequence may be a suitable promoter sequence, a nucleic acid sequence recognized by a Bacillus cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the biological substance. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the Bacillus cell of choice, obtainable from a gene that directs the synthesis of biological material extracellularly or intracellularly, homologous to the Bacillus cell, or heterologous. Examples of suitable promoters directing transcription of the nucleic acid constructs of the invention, particularly in Bacillus cells, are those derived from the lac operon of Escherichia coli (E.coli), Streptomyces coelicolor (Streptomyces coelicolor) agarase gene (dagA ), Bacillus subtilis fructan sucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene ( amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and promoters obtained from prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75 : 3727-3731), and the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA80: 21-25). More promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a Bacillus cell to terminate transcription. A terminator sequence is operably linked to the 3' end of the nucleic acid sequence directing the synthesis of biological material. Any terminator that is functional in the Bacillus cell of choice may be used in the present invention.

The regulatory sequence may also be a suitable leader sequence, the untranslated region of an mRNA important for translation by Bacillus cells. A leader sequence is operably linked to the 5' end of a nucleic acid sequence that directs the synthesis of biological material. Any leader sequence that is functional in the Bacillus cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the polypeptide that directs the expressed polypeptide into the cell's secretory pathway. The signal peptide coding region may be the native sequence of the polypeptide or may be obtained from a heterologous source. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is heterologous to that portion of the coding sequence encoding the secreted polypeptide. Since coding sequences normally do not contain a signal peptide coding region, a heterologous signal peptide coding region may be necessary. Alternatively, the heterologous signal peptide coding region may replace only the native signal peptide coding region in order to enhance secretion of the polypeptide relative to the native signal peptide coding region normally associated with the coding sequence. The signal peptide coding region can be obtained from the amylase or protease gene of a Bacillus species. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of the Bacillus cell of choice may be used in the present invention.

The effective signal peptide coding region of Bacillus cells is the signal peptide coding region obtained from the following genes: Bacillus NCIB 11837 (maltogenic) amylase gene, Bacillus stearothermophilus alpha-amylase gene, Bacillus licheniformis subtilisin gene , Bacillus licheniformis β-lactamase gene, Bacillus stearothermophilus neutral protease gene (nprT, nprS, nprM), and Bacillus subtilis prsA gene. Simonen and Palva, 1993, Microbiological Reviews 57: 109-137 describe further signal peptides.

In the methods of the present invention, recombinant expression vectors containing nucleic acid sequences, promoters, and transcriptional and translational termination signals can be used for recombinant production of polypeptides or other biological substances. The above-mentioned various nucleic acids and regulatory sequences can be connected to prepare a recombinant expression vector, which can include one or more appropriate restriction enzyme sites, allowing the insertion of nucleic acid sequences that guide the synthesis of polypeptides or biological substances at these sites Or replace. Alternatively, the nucleic acid sequence can be expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into a suitable expression vector. In the resulting expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate expression control sequences, which can result in secretion.

A recombinant expression vector can be any vector that facilitates recombinant DNA procedures and that results in the expression of a nucleic acid sequence. The choice of vector always depends on the compatibility between the vector and the Bacillus cells into which it will be introduced. Vectors can be linear or closed circular plasmids. A vector may be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity whose replication is not affected by chromosomal replication, eg, a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. A vector may contain any means to ensure self-replication. Alternatively, when introduced into a Bacillus cell, the vector may be one that is integrated into the genome and replicates with the chromosome into which it has integrated. The vector system may be a single vector or plasmid, or two or more vectors or plasmids that together contain the entire DNA or transposon to be introduced into the genome of the Bacillus cell.

When introduced into a Bacillus cell, the vector can be integrated into the Bacillus cell genome. For integration, the vector may rely on nucleic acid sequences directing the synthesis of biological material, or any other element of the vector for stable integration of the vector into the genome by homologous recombination. Alternatively, the vector may contain additional nucleic acid sequences that direct integration into the genome of the Bacillus cell by homologous recombination. This additional nucleic acid sequence enables integration of the vector into the genome of the Bacillus cell at a precise location in the chromosome. To increase the likelihood of integration at precise locations, the integrating element should preferably contain a nucleic acid of sufficient number of nucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, most preferably 800 to 1,500 bases Base pairs, which are highly homologous to the corresponding target sequences to increase the likelihood of homologous recombination. The integrated element may be any sequence homologous to the target sequence in the genome of the Bacillus cell. Furthermore, integrated elements may be non-coding or coding nucleic acid sequences.

For autonomous replication, the vector may further contain an origin of replication enabling autonomous replication of the vector in the Bacillus cell under consideration. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which permit replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1, which permit replication in Bacillus. The origin of replication may be one having a mutation such that its function is temperature sensitive in Bacillus cells (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of the nucleic acid sequence directing the synthesis of the biological material of interest can be introduced into the Bacillus cell to amplify the expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the genome of the Bacillus cell and selecting transformants using methods well known in the art. A convenient method for obtaining amplification of genomic DNA sequences is described in WO 94/1496.

The vector preferably contains one or more selectable markers which allow easy selection of transformed cells. A selectable marker is a gene whose product confers traits such as insecticide resistance, heavy metal resistance, auxotrophic prototrophy, etc. Examples of bacterial selectable markers are the dal gene of B. subtilis or B. licheniformis, or markers that confer resistance to antibiotics such as ampicillin, kanamycin, erythromycin, chloramphenicol or tetracycline. Alternatively, selection can be performed by means of co-transformation, e.g., as described in WO 91/09129, with selectable markers on separate vectors.

Methods for linking the above elements to construct recombinant expression vectors are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

Transformants of Bacillus cells can be produced, for example, by protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, for example, Young and Spizize, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6:742- 751), or by conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278).

The present invention also relates to a method for obtaining a parental bacillus cell mutant, comprising: (a) introducing a modified first nucleic acid sequence containing at least one of the cypX and yvmC genes involved in the production of red pigment into the bacillus cell middle; and (b) identifying mutant cells containing the modified nucleic acid sequence from step (a), wherein the mutant cells do not produce red pigment.

The present invention further relates to a parental Bacillus cell mutant comprising a first nucleic acid sequence directing the synthesis of a heterologous biological substance and a second nucleic acid sequence comprising a modification of at least one of the cypX and yvmC genes involved in the production of a red pigment, Among them, the mutant cells produced less red pigment than the parental Bacillus cells when cultured under the same conditions.

The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

Example

All primers and oligonucleotides were provided by MWG Biotech, Inc., High Point, NC.

Bacillus subtilis strains were made competent using the method described by Anagnostopoulos and Spizizen, 1961, Journal of Bacteriology 81: 741-746.

DNA sequencing was performed with an ABI 3700 sequencer (Sequencing) (Applied Biosystems, Inc., Foster City, CA).

Example 1: Identification of cypX-yvmC and yvmB-yvmA operons using DNA microarrays

Bacillus subtilis RB 128 strain is Bacillus subtilis A164Δ5 strain (Bacillus subtilis ATCC 6051A with spoIIAC, aprE, nprE, amyE, and srfC genes deleted) obtained according to the method of US Patent No. 5,891,701. The Bacillus subtilis strain RB128 contains a heterologous gene encoding a Bacillus maltogenic amylase. Bacillus subtilis BRG1 strain was obtained by N-methyl-N'nitrosoguanidine (NTG) mutagenesis of Bacillus subtilis RB128 strain according to the following protocol. Treatment with three different concentrations of N-methyl-N'nitrosoguanidine (NTG): 0.26 mg/ml, 0.53 mg/ml and 1.06 mg/ml produced 98.2%, 99.5% and 99.9% kill, respectively Bacillus subtilis RB128 strain cells grown to logarithmic phase. 100 microliters of cells per treatment group were overgrown to 6-fold in 1 ml wells of a 24-well plate. Growth halos were preserved in 10% glycerol and frozen at -80°C. The library sizes were approximately 15500, 4200 and 250 mutants for each treatment, respectively. Isolate the Bacillus subtilis BRG1 mutant from the Bacillus subtilis RB128 strain treated with 0.26mg/ml NTG, and cultivate BRG148 hours in 1.5 liters of medium with pH 7±0.2 at 40-41°C, wherein each liter of medium consists of 50 g Hydrolyzed protein, 6.5 g KH ₂ PO ₄ , 4.5 g Na ₂ HPO ₄ , 3.0 g (NH ₄ ) ₂ SO ₄ , 2.0 g sodium citrate-2H ₂ O, 3.0 g MgSO ₄ , 0.15 mg biotin, 0.5 g CaCl ₂ -2H ₂ O, and trace metals. Sugar was supplied to the fermentation at a maximum rate of 8 grams per liter per hour. The culture was sparged with air at a rate of 1 to 2 liters per minute and the culture was agitated at 1300 rpm. The color of the whole broth of the Bacillus subtilis BRG1 strain was light brown in contrast to the dark brown color of the whole broth of the Bacillus subtilis RB128 strain. A red pigment was seen in the cell pellet of the whole Bacillus subtilis strain RB128 broth, whereas no red pigment was observed in the cell pellet of the Bacillus subtilis strain BRG1.

Total cellular RNA was obtained from 6, 12, 24, 29 and 46 hour fermentation samples (10 ml) of B. subtilis strains RB 128 and BRG1. RNA was obtained from cell pellets prepared from fermentation samples stored at -80°C. For RNA preparation, frozen cell pellets were resuspended in 1 ml of diethylpyrocarbonate (DEPC)-treated water and nine replicates were prepared using the Fast RNA Blue kit (Bio101, Inc., Vista, CA). Replicates are then pooled into one tube for cDNA probe preparation.

10 replicate cDNA targets per time point were prepared and linked to ORFs of B. PCR fragment microarray hybridization. According to the method of Eisen and Burn, 1999, Methods in Enzymology 303:179-205, use Cy5 (Amersham Corporation, Arlington Heights, IL) to mark the cDNA of Bacillus subtilis RB 128 strain, and use Cy3 (Amersham Corporation, Arlington Heights, IL) ) to mark the cDNA of the Bacillus subtilis BRG1 strain. Cy3 (a green fluorescent dye) and Cy5 (red fluorescent dye) reporters were detected with solid-state lasers operating at 532 nm and 632 nm, respectively. Arrays were scanned with QuantArray (PerkinElmer Lifesciences, Inc., Boston, MA) and formatted for analysis, and data were imported into GeneSpring (Silicon Genetics, Inc., Redwood City, CA) for final analysis. Replicated slides were analyzed for statistical significance using the SAM Excel add-in from Stanford University (Tusher et al., 2001, Proceedings of the National Academy of Sciences USA 98:5116-5121). The cypX-yvmC and yvmB-yvmA operons were identified as potential sites involved in the formation of the red pigment, pulcemin (cypX: Figure 1, SEQ ID NOs: 1 and 2, accession number BG12580; yvmC: Figure 2, SEQ ID NOs: 3 and 4, accession number BG14121; yvmB: Figure 3, SEQ ID NOs: 5 and 6, accession number BG11018; and yvmA: Figure 4, SEQ ID NOs: 7 and 8, accession number BG14120). The cypX-yvmC and yvmB-yvmA operons were consistently downregulated in the B. subtilis BRG1 strain compared to the B. subtilis RB 128 strain at the 12-46 h time point.

A second microarray experiment was performed using two replicated cDNA targets hybridized to the B. subtilis ORFs oligonucleotide microarray. Oligonucleotides were purchased from Compugen, Inc., Jamesburg, NJ, and printed at a concentration of 10 μM on poly-L-lysine-coated glass slides according to the method described by Berka et al., 2002, supra, Create a density of 4 B. subtilis genomes per slide. The cDNAs of Bacillus subtilis RB128 and BRG1 strains were labeled according to the method described above. Arrays were scanned and formatted for analysis using a GenePix 4000B scanner and GenePix Pro version 4.1 software (Axon Instruments, Inc., Union City, CA). The replicated genomes were analyzed for statistical significance using the SAM Excel add-in described above, and genes identified as significant were imported into GeneSpring version 4.2. In this second microarray experiment, only the cypX-yvmC operon was identified as a potential site involved in red pigmentation.

Embodiment 2: Construction of Bacillus subtilis MaTa17 strain

Using primers 1 and 2, the cypX-yvmC and yvmB-yvmA operons were PCR amplified from Bacillus subtilis strain BRG1 as single fragments.

Primer 1: 5'-CATGGGAGAGACCTTTGG-3' (SEQ ID NO: 9)

Primer 2: 5'-GTCGGTCTTCCATTTGC-3' (SEQ ID NO: 10)

The amplification reaction (50 μl) consisted of: 200 ng of Bacillus subtilis strain BRG1 chromosomal DNA, 0.4 μM each of primers 1 and 2, 200 μM each of dATP, dCTP, dGTP, and dTTP, _1X Expand ^™ containing 1.5 mM MgCl High Fidelity buffer, and 2.6 units of Expand ^™ High Fidelity PCR System Enzyme Mix (Roche Diagnostic Corporation, Indianapolis, IN). According to the manufacturer's requirements (Genomic DNA Handbook, QIAGEN, Inc., Valencia, CA, 1999-2001, pp.38-47), use QIAGEN tip-20 column (QIAGEN, Inc., Valencia, CA) to obtain Bacillus subtilis Chromosomal DNA of BRG1 strain. The amplification reaction was carried out in a RoboCycler 40 thermocycler (Stratagene, Inc, La Jolla, CA), and the reaction program was: 1 cycle, 95°C for 3 minutes; 10 cycles, each cycle at 95°C for 1 minute, 58°C for 1 minute. minutes, and 4 minutes at 68°C; 20 cycles of 95°C for 1 minute, 58°C for 1 minute, and 68°C for 4 minutes and 20 seconds; followed by 1 cycle of 72°C for 7 minutes. The reaction products were analyzed by agarose gel electrophoresis using a 0.8% agarose-25 mM Tris base-25 mM borate-0.5 mM disodium edetate buffer (0.5X TBE) gel.

The resulting fragment containing the cypX-yvmC and yvmB-yvmA operons was cloned into pCR2.1 using the TA-TOPO cloning kit according to the manufacturer's instructions (Invitrogen, Inc., Carlsbad, CA), and transformed into Escherichia coli (E. coli) in OneShot ^™ cells. Transformants were selected on yeast-tryptone (2X YT) agar plates supplemented with 100 μg ampicillin per ml. Plasmid DNA was isolated from several transformants using a QIAGEN tip-20 column according to the manufacturer's instructions and confirmed by DNA sequencing using the M13(-20) forward primer, M13 reverse primer, and primers 3-18 indicated below . M13(-20) forward primer and M13 reverse primer were obtained from Invitrogen, Inc, Carlsbad, CA. The resulting plasmid was designated pMRT084 (Figure 5).

Primer 3: 5'-CGACCACTGTATCTTGG-3' (SEQ ID NO: 11)

Primer 4: 5'-GAGATGCCAAACAGTGC-3' (SEQ ID NO: 12)

Primer 5: 5'-CATGTCCATCGTGACG-3' (SEQ ID NO: 13)

Primer 6: 5'-CAGGAGCATTTGATACG-3' (SEQ ID NO: 14)

Primer 7: 5'-CCTTCAGATGTGATCC-3' (SEQ ID NO: 15)

Primer 8: 5'-GTGTTGACGTCAACTGC-3' (SEQ ID NO: 16)

Primer 9: 5'-GTTCAGCCTTTCCTCTCG-3' (SEQ ID NO: 17)

Primer 10: 5'-GCTACCTTCTTTCTTAGG-3' (SEQ ID NO: 18)

Primer 11: 5'-CGTCAATATGATCTGTGC-3' (SEQ ID NO: 19)

Primer 12: 5'-GGAAAGAAGGTCTGTGC-3' (SEQ ID NO: 20)

Primer 13: 5'-CAGCTATCAGCTGACAG-3' (SEQ ID NO: 21)

Primer 14: 5'-GCTCAGCTATGACATATTCC-3' (SEQ ID NO: 22)

Primer 15: 5'-GATCGTCTTGATTACCG-3' (SEQ ID NO: 23)

Primer 16: 5'-AGCTTTATCGGTGACG-3' (SEQ ID NO: 24)

Primer 17: 5'-TGAGCACGATTGCAGG-3' (SEQ ID NO: 25)

Primer 18: 5'-CATTGCGGAGACATTGC-3' (SEQ ID NO: 26)

DNA between the cypX-yvmC and yvmB-yvmA operons and the published Bacillus subtilis 168 sequence (Kunst et al., 1997, Nature 390:249-256) amplified from Bacillus subtilis BRG1 and cloned into plasmid pMRT084 Sequence comparison showed that these sequences were identical. To generate a B. subtilis strain with these operons deleted, plasmid pMRT084 was digested with BsgI to delete most of the cypX-yvmC and yvmB-yvmA operons, leaving approximately 500 bases at each end. Digested BsgI DNA was treated with T4 DNA polymerase and shrimp alkaline phosphatase (SAP) according to the manufacturer's instructions (Roche Diagnostics Corporation, Indianapolis, IN). Plasmid pECC1 (Youngman et al., 1984, Plasmid 12:1-9) was digested with Smal. A fragment of approximately 5100 bp and a fragment containing the chloramphenicol resistance gene ( cat), the two fragments were ligated, and E. coli (E. coli) XL1Blue cells were transformed according to the manufacturer's (Stratagene, Inc., La Jolla, CA) requirements. Transformants were selected on 2X YT agar plates supplemented with 100 μg ampicillin per ml. Transformants carrying the correct plasmids with most of the cypX-yvmC and yvmB-yvmA operons deleted were identified by PCR amplification using primers 19 and 20. PCR amplification was carried out in a 50 μl reaction solution consisting of 1 ng of plasmid DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, and 1X PCR buffer _II containing 2.5 mM MgCl (Applied Biosystems, Inc., Foster City, CA), and 2.5 units of AmpliTaq Gold ^™ DNA polymerase (Applied Biosystems, Inc., Foster City, CA). The reaction was carried out in a RoboCycler 40 thermal cycler, and the reaction program was: 95°C for 10 minutes, 1 cycle; 25 cycles, each cycle was: 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute; and 72°C 7 minutes, 1 cycle. PCR products were visualized using a 0.8% agarose-0.5X TBE gel. This construct was called pMRT086 (Figure 6).

Primer 19: 5'-TAGACAATTGGAAGAGAAAAGAGATA-3' (SEQ ID NO: 27)

Primer 20: 5'-CCGTCGCTATTGTAACCAGT-3' (SEQ ID NO: 28)

Plasmid pMRT086 was linearized with ScaI and transformed into B. subtilis RB128 competent cells at 0.2 μg chloramphenicol per ml. Transformants were selected on tryptone blood agar base (TBAB) plates containing 5 μg chloramphenicol per ml and grown at 37°C for 16 hours. Chromosomal DNA was prepared from several transformants using QIAGEN tip-20 columns according to the manufacturer's instructions. Chloramphenicol resistant colonies with PCR deletion of the cypX-yvmC and yvmB-yvmA operons were screened by PCR using primers 3 and 19, 3 and 20, 4 and 19, and 3 and 20. Perform PCR amplification in a 50 μl reaction consisting of 200 ng of chromosomal DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1X PCR buffer II containing 2.5 mM MgCl ₂ , and 2.5 units AmpliTaq Gold ^™ DNA polymerase composition. The reaction was carried out in a RoboCycler 40 thermal cycler, and the reaction program was: 95°C for 10 minutes, 1 cycle; 25 cycles, each cycle was: 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute; and 72°C 7 minutes, 1 cycle. PCR products were visualized using a 0.8% agarose-0.5X TBE gel. The resulting strain of Bacillus subtilis RB128 with deletion of cypX-yvmC and yvmB-yvmA was named Bacillus subtilis MaTa17.

Fermentation of Bacillus subtilis MaTal7 was carried out using the same medium and conditions as described in Example 1 . After 48 hours, the Bacillus subtilis MaTa17 strain produced no observable red pigment. Furthermore, a second DNA microarray analysis in Example 1 identified the cypX-yvmC operon as the only operon involved in red pigment synthesis, whereas Examples 3 and 4 below show that deletion of the cypX or yvmC genes is essential for red pigment The elimination of is required. Therefore, in order to test the elimination of red pigment useful, in various Bacillus subtilis strains, such as Bacillus subtilis A164Δ5 (U.S. Patent No. 5,891,701), Bacillus subtilis RB194 and RB194RB197 (WO 03/054163), and others described herein Deletion of the cypX gene in the Bacillus subtilis strain, the elimination of the red pigment will benefit the recovery of the product.

Example 3: Construction of Bacillus subtilis A164Δ5ΔcypX strain

To demonstrate the role of the cypX gene in red pigment synthesis, the cypX gene was deleted from Bacillus subtilis A164Δ5 (US Patent No. 5,891,701). Bacillus subtilis A164Δ5 competent cells were transformed using plasmid pMRT122 (WO03/054163). Transformants were selected on TBAB-agar plates supplemented with 1 μg erythromycin and 25 μg lincomycin per ml and cultured at 30°C for 24-48 hours. The deleted cypX gene was introduced into the chromosome of B. subtilis A164Δ5 by Campbell-type integration, and B. subtilis was selected for production by incubating freshly streaked B. subtilis A164Δ5 (pMRT122) cell plates at 45°C for 16 hours Healthy growing colonies of strain A164Δ5::pMRT122. Several healthy growing colonies were inoculated into 1 ml of LB broth medium and cultured overnight at 30 °C, 250 rpm. Use 10 μl of cultured cells for at least 3 consecutive passages. After the last passage, cultured cells were streaked on LB agar plates and incubated at 37°C for 16 hours. A single colony was picked and inoculated in duplicate on LB agar plates and TBAB plates supplemented with 1 μg erythromycin and 25 μg lincomycin per ml, and grown at 37°C for 16 hours. Using REDextract-N-Amp ^TM Plant PCR Kit (Sigma Chemical Company, St.Louis, MO), the chromosomal DNA of potential integrants was isolated according to the following steps: a single Bacillus colony was inoculated into 100 μl extract (Sigma Chemical Company, St. Louis, MO). Louis, MO) at 95°C for 10 minutes and then diluted with an equal volume of diluent (Sigma Chemical Company, St. Louis, MO). PCR was performed using 4 μl of extracted DNA with REDextract-N-Amp PCR reaction mix and primers 12 and 21 according to the manufacturer's instructions. Perform PCR reaction in RoboCycler 40 thermal cycler, the reaction program is: 9 minutes at 95°C, 1 cycle; 3 cycles, each cycle is: 1 minute at 95°C, 1 minute at 52°C, 1 minute at 72°C; 27 cycles Cycle, each cycle is: 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute; and 72°C for 5 minutes, 1 cycle. PCR products were visualized in a 0.8% agarose-0.5X TBE gel. The resulting strain was called Bacillus subtilis A164Δ5ΔcypX. Spizizen's Basic Salt-Agar (SMS) plates (Anagnostopoulos and Spizizen , 1961, supra) showed the presence or loss of red pigment in Bacillus subtilis. Bacillus subtilis A164Δ5ΔcypX appears to be colorless when compared to Bacillus subtilis A164Δ5.

Primer 21: 5'-CATGGGAGAGACCTTTGG-3' (SEQ ID NO: 29)

Example 4: Construction of Bacillus subtilis A164Δ5ΔyvmC strain

To verify whether cypX and/or yvmC are responsible for red pigment synthesis, the yvmC gene was deleted, leaving the cypX gene intact. Plasmids pMRT074 (WO 03/054163) and pMRT084 were digested with EcoRI and HindIII. Using the QIAquick DNA Purification Kit, according to the manufacturer's requirements, isolate the approximately 4300bp fragment of pMRT074 and the approximately 1700bp fragment of pMRT084 from a 2% agarose-0.5X TBE gel, ligate the two fragments, and use for transformation Bacillus subtilis 168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 µg erythromycin and 25 µg lincomycin per ml and incubated at 30°C for 24 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5X TBE gel by restriction analysis with Dral. The resulting construct was called pMRT126 (Figure 7).

Plasmid pMRT126 was digested with Ecl136II/Eco47III to delete the yvmC gene, and the digested fragments were ligated and used to transform Bacillus subtilis A168Δ4. Transformants were selected on TBAB-agar plates supplemented with 1 µg erythromycin and 25 µg lincomycin per ml and incubated at 30°C for 24 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5X TBE gel by restriction analysis with Dral. The resulting plasmid was designated pMRT128 (Figure 8).

Plasmid pMRT128 was used to transform Bacillus subtilis A164Δ5 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 µg erythromycin and 25 µg lincomycin per ml and incubated at 30°C for 24-48 hours. The deleted yvmC gene was introduced into the chromosome of Bacillus subtilis 164Δ5 by Campbell-type integration and healthy growing colonies were selected by incubating freshly streaked Bacillus subtilis A164Δ5 (pMRT128) cell plates at 45°C for 16 hours . Inoculate several healthy growing colonies into 1 ml of LB broth medium and culture overnight at 30°C, 250rpm. Use 10 μl of cultured cells for at least 3 consecutive passages. After the last passage, cultured cells were streaked on LB agar plates and incubated at 37°C for 16 hours. Pick a single colony and inoculate in duplicate on LB agar plates, TBAB plates supplemented with 1 μg erythromycin and 25 μg lincomycin per ml, and SMS plates containing trace metals described in Example 3, at 37 Grow for 16-48 hours at °C. Chromosomal DNA from erythromycin-susceptible colonies was isolated using the REDextract-N-Amp ^™ Plant PCR Kit (Sigma Chemical Company, St. Louis, MO) as described in Example 3, using the PCR cycle described in Example 3 Conditions, with primers 7 and 10, were screened for deleted yvmC genes by PCR. PCR products were visualized in a 0.8% agarose-0.5X TBE gel. The presence or absence of the red pigment in Bacillus subtilis was shown on Spizizen's minimal salt-agar (SMS) plates containing trace metals. The yvmC-deleted strain, which appeared to be colorless when compared with the wild-type strain, was designated Bacillus subtilis A164Δ5ΔyvmC.

Example 5: Fermentation of Bacillus subtilis strains

Bacillus subtilis RB187, RB194 and RB197 strains were constructed according to the method described in WO 03/054163 and cultured in 3 liter fermentors containing 1.5 liters of minimal salt medium consisting of 6.5 g of _KH2PO per liter _4. 4.5 g Na ₂ HPO ₄ , 3.0 g (NH ₄ ) ₂ SO ₄ , 2.0 g sodium citrate, 3.0 g MgSO ₄ ·7H ₂ O, 0.15 g biotin, 15 g sugar, 0.5 g CaCl ₂ ·2H ₂ O and trace element composition. Sugar was supplied to the fermentation at a rate of 2 grams of sugar per liter per hour. The culture was sparged with air at a rate of 1 to 2 liters per minute and the culture was agitated at 1250 rpm. Fermentation was maintained at pH 7.0±0.2 and temperature 32-37°C. Carry out fermentation with Bacillus subtilis RB187 strain, through 12 hours, visible red pigment product in whole broth supernatant and cell pellet, strengthen the remainder of fermentation, still visible red pigment product until 48 hours. Fermentation with Bacillus subtilis RB194 and RB197 strains, no red pigment product was observed. Table 1 summarizes the results of red pigment synthesis of the strains evaluated in the present invention.

Table 1. Summary of red pigment synthesis by strains evaluated

Example 6: Construction of Bacillus licheniformis SJ1904ΔcypX strain

Primers 22 and 23 were used to PCR amplify the cypX gene of Bacillus licheniformis SJ1904 (US Patent No. 5,733,753).

Primer 22: 5'-GAATTCGCAGGAGGAACGAGTATG-3' (SEQ ID NO: 30)

Primer 23: 5'-AAGCTTGAAGATCAGTGAGGCAGC-3' (SEQ ID NO: 31)

The amplification reaction (50 μl) consisted of 200 ng of Bacillus licheniformis SJ1904 chromosomal DNA, 0.4 μM each of primers 22 and 23, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1X Expand ^™ high-fidelity buffer containing 1.5 mM MgCl ₂ , and 2.6 units of Expand ^(TM) High-Fidelity PCR System Enzyme Mix (Roche Diagnostic Corporation, Indianapolis, IN) were made up. Chromosomal DNA of Bacillus licheniformis SJ1904 was obtained using a QIAGEN tip-20 column according to the manufacturer's instructions. The amplification reaction was carried out in a RoboCycler 40 thermal cycler, and the reaction program was: 94°C for 2 minutes, 1 cycle; 30 cycles, each cycle was: 94°C for 1 minute, 52°C for 1 minute, 68°C for 2 minutes; then 72°C for 7 minutes, 1 cycle. The reaction products were analyzed by agarose gel electrophoresis using 0.8% agarose-0.5X TBE gel. According to the manufacturer's requirements, the obtained fragment (about 1300 bp) containing the cypX gene was cloned into pCR2.1 using the TA-TOPO cloning kit, and used to transform Escherichia coli (E.coli) OneShot ^™ cells. Transformants were selected on 2X YT agar plates supplemented with 100 μg ampicillin per ml. Plasmid DNA of several transformants was isolated using a QIAGEN tip-20 column and verified by DNA sequencing using the M13(-20) forward primer and the M13 reverse primer according to the manufacturer's instructions. The resulting plasmid was designated pMRT121 (Fig. 21).

Plasmid pMRT121 was digested with NruI and PmlI to delete the cypX gene, leaving about 350 bp at each end, and the two fragments were ligated and used to transform E. coli XL1Blue cells according to the manufacturer's requirements. Transformants were selected on 2X YT agar plates supplemented with 100 μg ampicillin per ml and incubated at 37°C for 16 hours. Transformants carrying the correct plasmid were identified on a 2% agarose-0.5XTBE gel by restriction analysis with Dral. The resulting construct was called pMRT123 (Figure 10).

Plasmids pMRT074 and pMRT123 were digested with EcoRI and HindIII. According to the manufacturer's instructions, use the QIAquick DNA purification kit to separate the approximately 700bp fragment of pMRT123 and the approximately 4300bp fragment of pMRT074 from 0.8% agarose-0.5X TBE gel, and connect the two fragments for transformation of Bacillus subtilis Bacillus A168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 µg erythromycin and 25 µg lincomycin per ml and incubated at 30°C for 24 hours. Transformants carrying the correct plasmid were identified on a 2% agarose-0.5X TBE gel by restriction analysis with Dral. The resulting construct was called pMRT124 (Figure 11).

The plasmid pMRT124 was used to transform B. licheniformis SJ1904 electrocompetent cells according to the method described by Xue et al., 1999, Journal of Microbiological Methods 34: 183-191. After electroporation, cells were cultured in LBSM medium (Luria-Bertani with 0.5M sorbose and 0.38M mannitol) supplemented with 0.2 μg/ml erythromycin for 2.5 to 3 hours, and cells were plated in 1 μg erythromycin and 25 μg lincomycin on a TBAB-agar plate, cultivated at 30° for 24-48 hours. The deleted cypX gene in plasmid pMRT124 was introduced into the chromosome of B. licheniformis (B. licheniformis) SJ1904 by Campbell-type integration by culturing freshly streaked B. licheniformis A164Δ5 (pMRT124) cell plates at 50°C For 16 hours, select healthy growing colonies. Inoculate several healthy growing colonies into 1 ml of LB broth medium and culture overnight at 30°C, 250rpm. Use 10 μl of cultured cells for at least 3 consecutive passages. After the last passage, cultured cells were streaked on LB agar plates and incubated at 37°C for 16 hours. A single colony was picked, inoculated in duplicate on LB agar plates and TBAB plates supplemented with 1 μg erythromycin and 25 μg lincomycin per ml, and grown at 37°C for 16 hours. Chromosomal DNA of erythromycin-susceptible colonies was isolated using the REDextract-N-Amp ^™ Plant PCR Kit as described in Example 3, and screened by PCR using the PCR cycling conditions described in Example 3 with primers 22 and 23 Deleted cypX gene. PCR products were visualized in a 0.8% agarose-0.5X TBE gel. The resulting strain was called Bacillus licheniformis SJ1904ΔcypX. The red color formed by B. licheniformis was shown by streaking B. licheniformis SJ1904 and B. licheniformis SJ1904ΔcypX together on Spizizen's minimal salt-agar (SMS) plates supplemented with trace metals (Example 3). Presence or loss of pigment. Plates were incubated at 37°C for 48 hours. The cypX-deleted strain appeared to be colorless when compared to the control strain, suggesting that the loss of red pigmentation was accomplished by deletion of the cypX gene.

Example 7: Isolation of red pigment from RB187 supernatant

The red pigment found in the broth medium of Bacillus subtilis strain RB187 was isolated by adjusting the pH of 40 ml of the supernatant to 1.5 with 6N HCl. The acidified broth was incubated at 94°C for 30 minutes and the pigment was precipitated by centrifugation at 2500 rpm for 20 minutes at 4°C in a SORVALL 6000B centrifuge (SORVALL, Inc., Newtown, CT). The red pigment was washed 3 times by centrifugation with 20 ml of HPLC grade water, dissolved in 10 ml of basic methanol, and passed through in the presence of excess ferric chloride according to the method described by Canale-Parola, 1963, Archiv für Mikrobiologie 46:414-427 Acidification adjusts the pH to 1.5 and recovers the red pigment. Spectral analysis of the red pigment from 600nm to 200nm in 2M NaOH yielded absorption spectra with peaks at 242nm, 280nm and 242nm. The UV-visible spectrum of this purified pigment is similar to the Puchemin absorption spectrum found by Canale-Parola. Solubility in alkaline methanol, insolubility in acid, and unique absorption spectrum together strongly suggest that the red pigment is Puqimin.

The invention described and claimed herein should not be limited in scope to the particular embodiments disclosed herein, as these are merely examples of several aspects of the invention. Any equivalent embodiments are intended to be considered within the scope of this invention. Indeed, various modifications of the invention, in addition to those herein shown and described, will be apparent to those skilled in the art from the foregoing description. Such modifications should also fall within the scope of the appended claims. To the extent of conflict, the present disclosure, including definitions, will control.

Various references are cited here, and the entire contents of the disclosures thereof are incorporated by reference.

sequence listing

<110> Novozymes Biotech (NOVOZYMES BIOTECH, INC.)

<120> Method for producing biological material in a pigment-deficient Bacillus cell mutant

<130>10302.200-WO

<150>US 60/398,853

<151>2002-07-26

<160>31

<170>PatentIn version 3.2

<210>1

<211>1215

<212>DNA

<213> Bacillus subtilis

<400>1

atgagccaat cgattaaatt gtttagtgtg ctttctgatc aatttcaaaa caatccatat 60

gcttattttt cacaactgcg ggaggaagat ccggttcatt atgaagagtc gatagacagt 120

tatttatca gccgctatca tgatgtccgc tatatccttc agcatccgga tatcttcacg 180

acgaaatcac ttgttgagcg tgccgaacca gtcatgcgag gccctgtgct ggcccaaatg 240

catggaaaag aacactctgc caaaagaaga attgtagtga gaagctttat cggtgacgca 300

ctggatcatc tgtctccatt gattaaacaa aatgcagaaa acttgttagc gccttatctt 360

gaaagaggga aaagtgatct cgtcaatgat tttggaaaga cgtttgcggt gtgcgtcacg 420

atggacatgc tcgggctgga taaaagagac catgaaaaaa tctctgagtg gcacagcgga 480

gttgccgatt ttatcacgag tatctctcaa tctcctgaag cgcgggcaca ttcgttatgg 540

tgcagcgaac agctttccca atacttgatg ccggtcatta aagaacgtcg cgtcaatccg 600

ggatcagatt taatttcgat cctatgtact tctgaatatg aaggcatggc gctgtcggac 660

aaggatatac tcgcactgat tcttaatgtg ctgttagccg caacggaacc ggctgataag 720

acgctggcac tgatgatcta ccatttgctc aacaatcctg agcagatgaa tgatgttttg 780

gctgaccgtt cgttagttcc gagagccatt gcggagacat tgcgttataa accgccggtt 840

cagctgattc cgcggcagct gtcccaagat acagtggtcg gcggtatgga aatcaaaaaa 900

gatacgattg ttttttgtat gatcggtgcg gctaaccggg accctgaagc atttgaacag 960

cctgacgtgt ttaatattca tcgggaagat cttggtatca agagcgcttt tagcggcgcc 1020

gcccggcatc tcgctttcgg atccggcatt cataactgtg taggagcagc ttttgccaaa 1080

aacgaaatcg aaattgtagc taatattgtg ctggataaga tgcggaatat cagattatagag 1140

gaagattttt gttatgctga gtccggtctg tatacacgcg gacctgtttc acttctcgtt 1200

gcgtttgacg gggca 1215

<210>

<211>405

<212>PRT

<213> Bacillus subtilis

<400>2

Met Ser Gln Ser Ile Lys Leu Phe Ser Val Leu Ser Asp Gln Phe Gln

1 5 10 15

Asn Asn Pro Tyr Ala Tyr Phe Ser Gln Leu Arg Glu Glu Asp Pro Val

20 25 30

His Tyr Glu Glu Ser Ile Asp Ser Tyr Phe Ile Ser Arg Tyr His Asp

35 40 45

Val Arg Tyr Ile Leu Gln His Pro Asp Ile Phe Thr Thr Lys Ser Leu

50 55 60

Val Glu Arg Ala Glu Pro Val Met Arg Gly Pro Val Leu Ala Gln Met

65 70 75 80

His Gly Lys Glu His Ser Ala Lys Arg Arg Ile Val Val Arg Ser Phe

85 90 95

Ile Gly Asp Ala Leu Asp His Leu Ser Pro Leu Ile Lys Gln Asn Ala

100 105 110

Glu Asn Leu Leu Ala Pro Tyr Leu Glu Arg Gly Lys Ser Asp Leu Val

115 120 125

Asn Asp Phe Gly Lys Thr Phe Ala Val Cys Val Thr Met Asp Met Leu

130 135 140

Gly Leu Asp Lys Arg Asp His Glu Lys Ile Ser Glu Trp His Ser Gly

145 150 155 160

Val Ala Asp Phe Ile Thr Ser Ile Ser Gln Ser Pro Glu Ala Arg Ala

165 170 175

His Ser Leu Trp Cys Ser Glu Gln Leu Ser Gln Tyr Leu Met Pro Val

180 185 190

Ile Lys Glu Arg Arg Val Asn Pro Gly Ser Asp Leu Ile Ser Ile Leu

195 200 205

Cys Thr Ser Glu Tyr Glu Gly Met Ala Leu Ser Asp Lys Asp Ile Leu

210 215 220

Ala Leu Ile Leu Asn Val Leu Leu Ala Ala Thr Glu Pro Ala Asp Lys

225 230 235 240

Thr Leu Ala Leu Met Ile Tyr His Leu Leu Asn Asn Pro Glu Gln Met

245 250 255

Asn Asp Val Leu Ala Asp Arg Ser Leu Val Pro Arg Ala Ile Ala Glu

260 265 270

Thr Leu Arg Tyr Lys Pro Pro Val Gln Leu Ile Pro Arg Gln Leu Ser

275 280 285

Gln Asp Thr Val Val Gly Gly Met Glu Ile Lys Lys Asp Thr Ile Val

290 295 300

Phe Cys Met Ile Gly Ala Ala Asn Arg Asp Pro Glu Ala Phe Glu Gln

305 310 315 320

Pro Asp Val Phe Asn Ile His Arg Glu Asp Leu Gly Ile Lys Ser Ala

325 330 335

Phe Ser Gly Ala Ala Arg His Leu Ala Phe Gly Ser Gly Ile His Asn

340 345 350

Cys Val Gly Ala Ala Phe Ala Lys Asn Glu Ile Glu Ile Val Ala Asn

355 360 365

Ile Val Leu Asp Lys Met Arg Asn Ile Arg Leu Glu Glu Asp Phe Cys

370 375 380

Tyr Ala Glu Ser Gly Leu Tyr Thr Arg Gly Pro Val Ser Leu Leu Val

385 390 395 400

Ala Phe Asp Gly Ala

405

<210>3

<211>1218

<212>DNA

<213> Bacillus subtilis

<400>3

gtgtacactt tggctcatac aaaatcaaag gcagtattga tcttatacac tgtttgcttc 60

agtgcatttt ttgcatcttt aagccagaac atttattcac ctattcttcc gatcattaaa 120

gaatcattcc atgtttccac agctatggtg aacctgtcag tctcagtttt tatgattgtg 180

acagcaataa tgcaaattat attaggagcg atcattgatt ttaaaggcgc tcggatcgtc 240

ttgattaccg gtattctggc aacggcagca gccagcatcg gctgtgcggt gactactgac 300

tttaccttgt ttctgatatt cagaatgata caggcagccg gttccgcagc actgcctctt 360

attgctgcca caacgatcgg acagctgttt acaggaaatg aacgcgggag tgcaatggga 420

acgtatcaaa tgctcctgtc tgtcgcaccg gctattgctc cagttctagg aggattcata 480

ggcggagcag ccggatacga agggattttt tggatacttg cggccatctc tatcgttttg 540

ctggtgacaa acagcatcac ctttcctaaa gattctccaa ctgaatctat gcagcaagcc 600

aaaggcaatg tgttcgctca ttataaatca atatttacaa atcgaacagg gaacgtcatt 660

ttgactttaa gttttgttct ctttttcatt tattttgcag taattgtcta cctcccaata 720

ttgctgacag agcattacca tatagatgtg ggtatagcag gactgttata tttgccgctg 780

gcgctgagca cgattgcagg tacgtttctg tttaaaagaa tacaggcaaa aatcgggctg 840

cacaccttgt ttatcggaag caatgtgatt gccgcctgca gcatcatttt atttgctgtt 900

aacacattccg tttctctcgt tctcatggct ctgacgctgg cactgtttgg catctcgatg 960

ggggttatc ctcccttgta ctctacaatg attackaatg aatttgagca caacagaggg 1020

agtgcaatcg gaatgtttaa ctttatccga tatacaggca tggcagcagg tccgatggta 1080

tctgcctact tgctcacaat gatgccgtct gccatgtcct ttagcctcct aggccttgga 1140

tttgccgcat tgagcttttg ccttcttccg ccaatgtttt cgccgcagaa gcgcacgaaa 1200

caaaaaaagc accacatg 1218

<210>4

<211>406

<212>PRT

<213> Bacillus subtilis

<400>4

Met Tyr Thr Leu Ala His Thr Lys Ser Lys Ala Val Leu Ile Leu Tyr

1 5 10 15

Thr Val Cys Phe Ser Ala Phe Phe Ala Ser Leu Ser Gln Asn Ile Tyr

20 25 30

Ser Pro Ile Leu Pro Ile Ile Lys Glu Ser Phe His Val Ser Thr Ala

35 40 45

Met Val Asn Leu Ser Val Ser Val Phe Met Ile Val Thr Ala Ile Met

50 55 60

Gln Ile Ile Leu Gly Ala Ile Ile Asp Phe Lys Gly Ala Arg Ile Val

65 70 75 80

Leu Ile Thr Gly Ile Leu Ala Thr Ala AlaAla Ser Ile Gly Cys Ala

85 90 95

Val Thr Thr Asp Phe Thr Leu Phe Leu Ile Phe Arg Met Ile Gln Ala

100 105 110

Ala Gly Ser Ala Ala Leu Pro Leu Ile Ala Ala Thr Thr Ile Gly Gln

115 120 125

Leu Phe Thr Gly Asn Glu Arg Gly Ser Ala Met Gly Thr Tyr Gln Met

130 135 140

Leu Leu Ser Val Ala Pro Ala Ile Ala Pro Val Leu Gly Gly Phe Ile

145 50 155 160

Gly Gly Ala Ala Gly Tyr Glu Gly Ile Phe Trp Ile Leu Ala Ala Ile

165 170 175

Ser Ile Val Leu Leu Val Thr Asn Ser Ile Thr Phe Pro Lys Asp Ser

180 185 190

Pro Thr Glu Ser Met Gln Gln Ala Lys Gly Asn Val Phe Ala His Tyr

195 200 205

Lys Ser Ile Phe Thr Asn Arg Thr Gly Asn Val Ile Leu Thr Leu Ser

210 215 220

Phe Val Leu Phe Phe Ile Tyr Phe Ala Val Ile Val Tyr Leu Pro Ile

225 230 235 240

Leu Leu Thr Glu His Tyr His Ile Asp Val Gly Ile Ala Gly Leu Leu

245 250 255

Tyr Leu Pro Leu Ala Leu Ser Thr Ile Ala Gly Thr Phe Leu Phe Lys

260 265 270

Arg Ile Gln Ala Lys Ile Gly Leu His Thr Leu Phe Ile Gly Ser Asn

275 280 285

Val Ile Ala Ala Cys Ser Ile Ile Leu Phe Ala Val Thr His Ser Val

290 295 300

Ser Leu Val Leu Met Ala Leu Thr Leu Ala Leu Phe Gly Ile Ser Met

305 310 315 320

Gly Val Ile Pro Pro Leu Tyr Ser Thr Met Ile Thr Asn Glu Phe Glu

325 330 335

His Asn Arg Gly Ser Ala Ile Gly Met Phe Asn Phe Ile Arg Tyr Thr

340 345 350

Gly Met Ala Ala Gly Pro Met Val Ser Ala Tyr Leu Leu Thr Met Met

355 360 365

Pro Ser Ala Met Ser Phe Ser Leu Leu Gly Leu Gly Phe Ala Ala Leu

370 375 380

Ser Phe Cys Leu Leu Pro Pro Met Phe Ser Pro Gln Lys Arg Thr Lys

385 390 395 400

Gln Lys Lys His His Met

405

<210>5

<211>507

<212>DNA

<213> Bacillus subtilis

<400>5

atgtctgatt tgacaaaaca gatgatatac gacatatacg tgagactgct gcaccttaat 60

gaacaaaaag cgaacacttc acttcagcaa ttttttaagg aggccgcaga agaggatgta 120

gctgaaattc ccaaaaatat gacaagcatt cacgtcattg actgcatcgg ccagcatgaa 180

cccattaata atgccggaat tgccagaaaa atgaacttat cgaaagcgaa tgtaacgaaa 240

atcagcacaa aactgatcaa ggaagaattc attaacagct atcagctgac agataacaaa 300

aaagaagttt attttaaatt aacccgtaaa ggcagacgga ttttcgactt acatgagaaa 360

ctgcataaaa aaaaggagct ggctttttac caattcctcg attcattttc acaagaagaa 420

caaaaggctg tattgaagtt tctagagcag ttgacgtcaa cacttgaagc agaacaaacc 480

gatgggactc cagacaaacc tgtaaag 507

<210>6

<211>169

<212>PRT

<213> Bacillus subtilis

<400>6

Met Ser Asp Leu Thr Lys Gln Met Ile Tyr Asp Ile Tyr Val Arg Leu

1 5 10 15

Leu His Leu Asn Glu Gln Lys Ala Asn Thr Ser Leu Gln Gln Phe Phe

20 25 30

Lys Glu Ala Ala Glu Glu Asp Val Ala Glu Ile Pro Lys Asn Met Thr

35 40 45

Ser Ile His Val Ile Asp Cys Ile Gly Gln His Glu Pro Ile Asn Asn

50 55 60

Ala Gly Ile Ala Arg Lys Met Asn Leu Ser Lys Ala Asn Val Thr Lys

65 70 75 80

Ile Ser Thr Lys Leu Ile Lys Glu Glu Phe Ile Asn Ser Tyr Gln Leu

85 90 95

Thr Asp Asn Lys Lys Glu Val Tyr Phe Lys Leu Thr Arg Lys Gly Arg

100 105 110

Arg Ile Phe Asp Leu His Glu Lys Leu His Lys Lys Lys Glu Leu Ala

115 120 125

Phe Tyr Gln Phe Leu Asp Ser Phe Ser Gln Glu Glu Gln Lys Ala Val

130 135 140

Leu Lys Phe Leu Glu Gln Leu Thr Ser Ser Thr Leu Glu Ala Glu Gln Thr

145 150 155 160

Asp Gly Thr Pro Asp Lys Pro Val Lys

165

<210>7

<211>753

<212>DNA

<213> Bacillus subtilis

<400>7

gtgaatgaga tgaccggaat ggtaacggaa agaaggtctg tgcattttat tgctgaggca 60

ttaacagaaa actgcagaga aatatttgaa cggcgcaggc atgttttggt ggggatcagc 120

ccatttaaca gcaggttttc agaggattat atttacagat taattggatg ggcgaaagct 180

caatttaaaa gcgtttcagt tttacttgca gggcatgagg cggctaatct tctagaagcg 240

cttggaactc cgagaggaaa ggctgaacga aaagtaagga aagaggtatc acgaaacagg 300

agarttgcag aaagagccct tgtggctcat ggcggggatc cgaaggcgat tcatacattt 360

tctgatttta tagataacaa agcctaccag ctgttgagac aagaagttga acatgcattt 420

tttgagcagc ctcattttcg acatgcttgt ttggacatgt ctcgtgaagc gataatcggg 480

cgtgcgcggg gcgtcagttt gatgatggaa gaagtcagtg aggatatgct gaatttggct 540

gtggaatatg tcatagctga gctgccgttt tttatcggag ctccggatat tttagaggtg 600

gaagagacac tccttgctta tcatcgtccg tggaagctgg gtgagaagat cagtaaccat 660

gaattttcta tttgtatgcg gccgaatcaa gggtatctca ttgtacagga aatggcgcag 720

atgctttctg agaaacggat cacatctgaa gga 753

<210>8

<211>251

<212>PRT

<213> Bacillus subtilis

<400>8

Met Asn Glu Met Thr Gly Met Val Thr Glu Arg Arg Ser Val His Phe

1 5 10 15

Ile Ala Glu Ala Leu Thr Glu Asn Cys Arg Glu Ile Phe Glu Arg Arg

20 25 30

Arg His Val Leu Val Gly Ile Ser Pro Phe Asn Ser Arg Phe Ser Glu

35 40 45

Asp Tyr Ile Tyr Arg Leu Ile Gly Trp Ala Lys Ala Gln Phe Lys Ser

50 55 60

Val Ser Val Leu Leu Ala Gly His Glu Ala Ala Asn Leu Leu Glu Ala

65 70 75 80

Leu Gly Thr Pro Arg Gly Lys Ala Glu Arg Lys Val Arg Lys Glu Val

85 90 95

Ser Arg Asn Arg Arg Phe Ala Glu Arg Ala Leu Val Ala His Gly Gly

100 105 110

Asp Pro Lys Ala Ile His Thr Phe Ser Asp Phe Ile Asp Asn Lys Ala

115 120 125

Tyr Gln Leu Leu Arg Gln Glu Val Glu His Ala Phe Phe Glu Gln Pro

130 135 140

His Phe Arg His Ala Cys Leu Asp Met Ser Arg Glu Ala Ile Ile Gly

145 150 155 160

Arg Ala Arg Gly Val Ser Leu Met Met Glu Glu Val Ser Glu Asp Met

165 170 175

Leu Asn Leu Ala Val Glu Tyr Val Ile Ala Glu Leu Pro Phe Phe Ile

180 185 190

Gly Ala Pro Asp Ile Leu Glu Val Glu Glu Thr Leu Leu Ala Tyr His

195 200 205

Arg Pro Trp Lys Leu Gly Glu Lys Ile Ser Asn His Glu Phe Ser Ile

210 215 220

Cys Met Arg Pro Asn Gln Gly Tyr Leu Ile Val Gln Glu Met Ala Gln

225 230 235 240

Met Leu Ser Glu Lys Arg Ile Thr Ser Glu Gly

245 250

<210>9

<211>18

<212>DNA

<213> Bacillus subtilis

<400>9

catgggag acctttgg 18

<210>10

<211>17

<212>DNA

<213> Bacillus subtilis

<400>10

gtcggtcttc catttgc 17

<210>11

<211>17

<212>DNA

<213> Bacillus subtilis

<400>11

cgaccactgt atcttgg 17

<210>12

<211>17

<212>DNA

<213> Bacillus subtilis

<400>12

gagatgccaa acagtgc 17

<210>13

<211>16

<212>DNA

<213> Bacillus subtilis

<400>13

catgtccatc gtgacg 16

<210>14

<211>17

<212>DNA

<213> Bacillus subtilis

<400>14

caggagcatt tgatacg 17

<210>15

<211>16

<212>DNA

<213> Bacillus subtilis

<400>15

ccttcagatg tgatcc 16

<210>16

<211>17

<212>DNA

<213> Bacillus subtilis

<400>16

gtgttgacgt caactgc 17

<210>17

<211>18

<212>DNA

<213> Bacillus subtilis

<400>17

gttcagcctt tcctctcg 18

<210>18

<211>18

<212>DNA

<213> Bacillus subtilis

<400>18

gctaccttct ttcttagg 18

<210>19

<211>18

<212>DNA

<213> Bacillus subtilis

<400>19

cgtcaatatg atctgtgc 18

<210>20

<211>17

<212>DNA

<213> Bacillus subtilis

<400>20

ggaaagaagg tctgtgc 17

<210>21

<211>17

<212>DNA

<213> Bacillus subtilis

<400>21

cagctatcag ctgacag 17

<210>22

<211>20

<212>DNA

<213> Bacillus subtilis

<400>22

gctcagctat gacatattcc 20

<210>23

<211>17

<212>DNA

<213> Bacillus subtilis

<400>23

gatcgtcttg attaccg 17

<210>24

<211>16

<212>DNA

<213> Bacillus subtilis

<400>24

agctttatcg gtgacg 16

<210>25

<211>16

<212>DNA

<213> Bacillus subtilis

<400>25

tgagcacga ttgcagg 16

<210>26

<211>17

<212>DNA

<213> Bacillus subtilis

<400>26

cattgcggag attgc 17

<210>27

<211>26

<212>DNA

<213> Bacillus subtilis

<400>27

tagacaattg gaagagaaaa gagata 26

<210>28

<211>20

<212>DNA

<213> Bacillus subtilis

<400>28

ccgtcgctat tgtaaccagt 20

<210>29

<211>18

<212>DNA

<213> Bacillus subtilis

<400>29

catgggag acctttgg 18

<210>30

<211>24

<212>DNA

<213> Bacillus licheniformis

<400>30

gaattcgcag gaggaacgag tatg 24

<210>31

<211>24

<212>DNA

<213> Bacillus licheniformis

<400>31

aagcttgaag atcagtgagg cagc 24

Claims (60)

1. A method of producing a heterologous biological substance, comprising: (a) cultivating a mutant of the parental Bacillus cell in a medium suitable for producing heterologous biological substances, wherein (i) the mutant cell contains a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence directing Synthesis of heterologous biological substances, the second nucleic acid sequence comprising a modification in at least one of the cypX and yvmC genes involved in red pigment production, and (ii) the mutant cells are the same as the parental Bacillus cells cultured under the same conditions In contrast, red pigment production is defective; and (b) recovering said heterologous biological material from said culture medium. 2. The method of claim 1, wherein at least one gene of the second nucleic acid sequence is cypX. 3. The method of claim 1, wherein at least one gene of the second nucleic acid sequence is yvmC. 4. The method of any one of claims 1-3, wherein the biological substance encoded by the first nucleic acid sequence is a biopolymer. 5. The method of claim 4, wherein the biopolymer is selected from nucleic acids, polyamides, polyamines, polyols, polypeptides or polysaccharides. 6. The method of claim 5, wherein the polypeptide is selected from the group consisting of antigens, growth factors, hormones, neurotransmitters, receptors, reporter proteins, structural proteins, or transcription factors. 7. The method of claim 5, wherein the polypeptide is an enzyme. 8. The method of claim 7, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. 9. The method of claim 5, wherein the polysaccharide is chitin, heparin, hyaluronan, or hyaluronic acid. 10. The method of any one of claims 1-3, wherein the biological substance encoded by the first nucleic acid sequence is a metabolite. 11. The method of claim 1, wherein the first nucleic acid sequence comprises a biosynthetic pathway or a metabolic pathway. 12. The method of any one of claims 1-3, wherein the mutant cell contains at least two copies of the first nucleic acid sequence directing the synthesis of biological material. 13. The method of any one of claims 1-3, wherein the Bacillus cells are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus Bacillus, Bacillus candidiasis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cells. 14. The method of any one of claims 1-3, wherein the Bacillus cell is a Bacillus subtilis cell. 15. The method of any one of claims 1-3, wherein the Bacillus cell is a Bacillus licheniformis cell. 16. The method of any one of claims 1-3, wherein the mutant cells produce at least 25% less red pigment than the parental Bacillus cells grown under the same conditions. 17. The method of any one of claims 1-3, wherein the mutant cells do not produce a detectable red pigment compared to the parental Bacillus cells grown under the same conditions. 18. The method of any one of claims 1-3, wherein the mutant cell further comprises a modification in one or more genes encoding proteases. 19. The method of claim 18, wherein the gene is nprE and/or aprE. 20. The method of any one of claims 1-3, wherein the mutant cell further comprises a modification in one or more genes selected from the group consisting of spoIIAC, srfA, srfB, srfC, srfD, or amyE. 21. A mutant of the parental Bacillus cell, containing a first nucleic acid sequence that directs the synthesis of heterologous biological substances and a second nucleic acid sequence that is involved in the production of red pigments in cypX and yvmC A modification is contained in at least one of the genes, wherein the mutant cells are defective in red pigment production compared to parental Bacillus cells grown under the same conditions. 22. The mutant of claim 21, wherein at least one gene of the second nucleic acid sequence is cypX. 23. The mutant of claim 21, wherein at least one gene of the second nucleic acid sequence is yvmC. 24. The mutant according to any one of claims 21-23, wherein the biological substance encoded by the first nucleic acid sequence is a biopolymer. 25. The mutant of claim 24, wherein the biopolymer is selected from nucleic acids, polyamides, polyamines, polyols, polypeptides or polysaccharides. 26. The mutant of claim 25, wherein the polypeptide is selected from an antigen, a growth factor, a hormone, a neurotransmitter, a receptor, a reporter protein, a structural protein, or a transcription factor. 27. The mutant of claim 25, wherein the polypeptide is an enzyme. 28. The mutant of claim 27, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. 29. The mutant of claim 25, wherein the polysaccharide is chitin, heparin, hyaluronan, or hyaluronic acid. 30. The mutant of any one of claims 21-23, wherein the biological substance encoded by the first nucleic acid sequence is a metabolite. 31. The mutant of claim 21, wherein the first nucleic acid sequence comprises a biosynthetic pathway or a metabolic pathway. 32. The mutant of any one of claims 21-23, which contains at least two copies of the first nucleic acid sequence directing the synthesis of biological material. 33. The mutant of any one of claims 21-23, wherein the Bacillus cell is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus, Bacillus cannula, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cells. 34. The mutant of any one of claims 21-23, which is a Bacillus subtilis cell. 35. The mutant of any one of claims 21-23, which is a Bacillus licheniformis cell. 36. The mutant of any one of claims 21-23, wherein the mutant cells produce at least 25% less red pigment than the parental Bacillus cells grown under the same conditions. 37. The mutant of any one of claims 21-23, wherein the mutant cells do not produce a detectable red pigment compared to the parental Bacillus cells cultured under the same conditions. 38. The mutant of any one of claims 21-23, further comprising a modification in one or more genes encoding proteases. 39. The mutant of claim 38, wherein the gene is nprE and/or aprE. 40. The mutant of any one of claims 21-23, further comprising a modification in one or more genes selected from spoIIAC, srfA, srfB, srfC, srfD, or amyE. 41. A method of obtaining a mutant of a parental Bacillus cell, comprising: (a) introducing a first nucleic acid sequence and a second nucleic acid sequence into the parental Bacillus cells, the first nucleic acid sequence directing the synthesis of heterologous biological substances, and the second nucleic acid sequence being involved in the production of red pigment in the cypX and yvmC genes contains the modifier in at least one of the ; and (b) identifying a mutant cell from step (a) containing the modified nucleic acid sequence, wherein the mutant cell is defective in red pigment production compared to a parental Bacillus cell cultured under the same conditions. 42. The method of claim 41, wherein at least one gene of the second nucleic acid sequence is cypX. 43. The method of claim 41, wherein at least one gene of the second nucleic acid sequence is yvmC. 44. The method of any one of claims 41-43, wherein the biological substance encoded by the first nucleic acid sequence is a biopolymer. 45. The method of claim 44, wherein the biopolymer is selected from nucleic acids, polyamides, polyamines, polyols, polypeptides or polysaccharides. 46. The method of claim 45, wherein the polypeptide is selected from the group consisting of antigens, growth factors, hormones, neurotransmitters, receptors, reporter proteins, structural proteins, or transcription factors. 47. The method of claim 45, wherein the polypeptide is an enzyme. 48. The method of claim 47, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. 49. The method of claim 45, wherein the polysaccharide is chitin, heparin, hyaluronan, or hyaluronic acid. 50. The method of any one of claims 41-43, wherein the biological substance encoded by the first nucleic acid sequence is a metabolite. 51. The method of claim 41, wherein the first nucleic acid sequence comprises a biosynthetic pathway or a metabolic pathway. 52. The method of any one of claims 41-43, wherein the mutant cell contains at least two copies of the first nucleic acid sequence directing the synthesis of biological material. 53. The method of any one of claims 41-43, wherein the Bacillus cells are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus Bacillus, Bacillus candidiasis, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cells. 54. The method of any one of claims 41-43, wherein the Bacillus cell is a Bacillus subtilis cell. 55. The method of any one of claims 41-43, wherein the Bacillus cell is a Bacillus licheniformis cell. 56. The method of any one of claims 41-43, wherein the mutant cells produce at least 25% less red pigment than the parental Bacillus cells grown under the same conditions. 57. The method of any one of claims 41-43, wherein the mutant cells do not produce a detectable red pigment compared to the parental Bacillus cells grown under the same conditions. 58. The method of any one of claims 41-43, wherein the mutant cell further comprises a modification in one or more genes encoding proteases. 59. The method of claim 58, wherein the gene is nprE and/or aprE. 60. The method of any one of claims 41-43, wherein the mutant cell further comprises a modification in one or more genes selected from the group consisting of spoIIAC, srfA, srfB, srfC, srfD, or amyE.

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